Polypeptide sequences of human EDG-1c

ABSTRACT

Human EDG-1c polypeptidees and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Human EDG-1c is identified as a selective receptor for sphingosine-1-phosphate (“S-1-P”) and for di-hydro S-1-P. Also disclosed are methods for discovering agonists and antagonists of the interaction between S-1-P and di-hydro S-1-P and their cellular receptor, human EDG-1c, which may have utility in the treatment of several human diseases and disorders, including, but not limited to the treatment of infections such as bacterial, fungal, protozoan and viral infections, particularly infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson&#39;s disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; stroke; congestive heart failure; left ventricular hypertrophy; arrythmias; restenosis after coronary artery angioplasty; vascular sclerosis; deleterious fibrosis; atherosclerosis; inflammation; angiogenesis; wound healing; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, delirium, dementia, and severe mental retardation; degenerative diseases, such as neurodegenerative diseases and ischemic stroke; and dyskinesias, such as Huntington&#39;s disease or Gilles dela Tourett&#39;s syndrome.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit to the earlier provisional U.S.application Ser. Nos. 60/077,369, filed on Mar. 9, 1998, and 60/087,102,filed on May 28, 1998, the contents of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polypeptides andpolynucleotides encoded by them and to the use of such polynucleotidesand polypeptides, and to their production. More particularly, thepolynucleotides and polypeptides of the present invention relate to theG-protein coupled receptors, hereinafter referred to as human EDG-1creceptor. This invention also relates to methods for discoveringagonists and antagonists of the interaction between sphingosine1-phosphate (hereinafter referred to as “S-1-P”) and di-hydrosphingosine 1-phosphate (also known as sphingoanine 1-phosphate andhereinafter referred to as “di-hydro S-1-P”) and their cellularreceptor, human EDG-1c receptor. The invention also relates to the useof human EDG-1c polynucleotides and polypeptides in therapy and inidentifying compounds which may be agonists, antagonists and/orinhibitors which are potentially useful in therapy, and to production ofsuch polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

[0003] The drug discovery process is currently undergoing a fundamentalrevolution as it embraces ‘functional genomics’, that is, highthroughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on ‘positional cloning’. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

[0004] Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available. There is a continuingneed to identify and characterize further genes and their relatedpolypeptides/proteins, as targets for drug discovery.

[0005] It is well established that many medically significant biologicalprocesses are mediated by proteins participating in signal transductionpathways that involve G-proteins and/or second messengers, e.g., cAMP(Lefkowitz, Nature, 1991, 351:353-354). Herein, these proteins arereferred to as proteins participating in pathways with G-proteins. Someexamples of these proteins include the G-protein coupled receptors, suchas those for adrenergic agents and dopamine (Kobilka, B. K., et al.,Proc. Natl Acad. Sci., USA, 1987, 84:46-50; Kobilka, B. K., et al.,Science, 1987, 238:650-656; Bunzow, J. R., et al., Nature, 1988,336:783-787), G-proteins themselves, effector proteins, e.g.,phospholipase C, adenyl cyclase, and phosphodiesterase, and actuatorproteins, e.g., protein kinase A and protein kinase C (Simon, M. I., etal., Science, 1991, 252:802-8).

[0006] For example, in one form of signal transduction, the effect ofhormone binding is activation of the enzyme, adenylate cyclase, insidethe cell. Enzyme activation by hormones is dependent on the presence ofthe nucleotide GTP. GTP also influences hormone binding. A G-proteinconnects the hormone receptor to adenylate cyclase. G-protein was shownto exchange GTP for bound GDP when activated by a hormone receptor. TheGTP-carrying form then binds to activated adenylate cyclase. Hydrolysisof GTP to GDP, catalyzed by the G-protein itself, returns the G-proteinto its basal, inactive form. Thus, the G-protein serves a dual role, asan intermediate that relays the signal from receptor to effector, and asa clock that controls the duration of the signal.

[0007] The membrane protein gene superfamily of G-protein coupledreceptors has been characterized as having seven putative transmembranedomains. The domains are believed to represent transmembrane a-helicesconnected by extracellular or cytoplasmic loops. G-protein coupledreceptors include a wide range of biologically active receptors, such ashormone, viral, growth factor and neuroreceptors.

[0008] G-protein coupled receptors (otherwise known as 7TM receptors)have been characterized as including these seven conserved hydrophobicstretches of about 20 to 30 amino acids, connecting at least eightdivergent hydrophilic loops. The G-protein family of coupled receptorsincludes dopamine receptors which bind to neuroleptic drugs used fortreating psychotic and neurological disorders. Other examples of membersof this family include, but are not limited to, calcitonin, adrenergic,endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin,histamine, thrombin, kinin, follicle stimulating hormone, opsins,endothelial differentiation gene-1, rhodopsins, odorant, andcytomegalovirus receptors.

[0009] Most G-protein coupled receptors have single conserved cysteineresidues in each of the first two extracellular loops which formdisulfide bonds that are believed to stabilize functional proteinstructure. The 7 transmembrane regions are designated as TM1, TM2, TM3,TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.

[0010] Phosphorylation and lipidation (palmitylation or farnesylation)of cysteine residues can influence signal transduction of some G-proteincoupled receptors. Most G-protein coupled receptors contain potentialphosphorylation sites within the third cytoplasmic loop and/or thecarboxy terminus. For several G-protein coupled receptors, such as theb-adrenoreceptor, phosphorylation by protein kinase A and/or specificreceptor kinases mediates receptor desensitization.

[0011] For some receptors, the ligand binding sites of G-protein coupledreceptors are believed to comprise hydrophilic sockets formed by severalG-protein coupled receptor transmembrane domains, said socket beingsurrounded by hydrophobic residues of the G-protein coupled receptors.The hydrophilic side of each G-protein coupled receptor transmembranehelix is postulated to face inward and form polar ligand binding site.TM3 has been implicated in several G-protein coupled receptors as havinga ligand binding site, such as the TM3 aspartate residue. TM5 serines, aTM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are alsoimplicated in ligand binding.

[0012] G-protein coupled receptors can be intracellularly coupled byheterotrimeric G-proteins to various intracellular enzymes, ion channelsand transporters (see, Johnson, et al., Endoc. Rev., 1989, 10:317-331)Different G-protein a-subunits preferentially stimulate particulareffectors to modulate various biological functions in a cell.Phosphorylation of cytoplasmic residues of G-protein coupled receptorshave been identified as an important mechanism for the regulation ofG-protein coupling of some G-protein coupled receptors. G-proteincoupled receptors are found in numerous sites within a mammalian host.

[0013] Over the past 15 years, nearly 350 therapeutic agents targeting 7transmembrane (7TM) receptors have been successfully introduced onto themarket.

SUMMARY OF THE INVENTION

[0014] In one aspect, the invention relates human EDG-1c polypeptidesand recombinant materials and methods for their production. Anotheraspect of the invention relates to methods for using such human EDG-1cpolypeptides and polynucleotides. Such uses include the treatment ofinfections such as bacterial, fungal, protozoan and viral infections,particularly infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson'sdisease; acute heart failure; hypotension; hypertension; urinaryretention; osteoporosis; angina pectoris; myocardial infarction; stroke;congestive heart failure; left ventricular hypertrophy; arrythmias;restenosis after coronary artery angioplasty; vascular sclerosis;deleterious fibrosis; atherosclerosis; inflammation; angiogenesis; woundhealing; ulcers; asthma; allergies; benign prostatic hypertrophy;migraine; vomiting; psychotic and neurological disorders, includinganxiety, schizophrenia, manic depression, depression, delirium,dementia, and severe mental retardation; degenerative diseases, such asneurodegenerative diseases and ischemic stroke; and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome, among others.

[0015] In accordance with another aspect of the present invention thereare provided methods of screening for compounds which bind to andactivate (agonist) or inhibit activation (antagonist) of human EDG-1cpolypeptides (receptors), and for their ligands.

[0016] In particular, the preferred method for identifying agonist orantagonist of a human EDG-1c polypeptide comprises:

[0017] (a) contacting a cell expressing on the surface thereof thepolypeptide, said polypeptide being associated with a second componentcapable of providing a detectable signal in response to the binding of acompound to said polypeptide, with a compound to be screened underconditions to permit binding to the polypeptide; and

[0018] (b) determining whether the compound binds to and activates orinhibits the polypeptide by measuring the level of a signal generatedfrom the interaction of the compound with the polypeptide.

[0019] In a further preferred embodiment, the method further comprisesconducting the identification of agonist or antagonist in the presenceof labeled or unlabeled S-1-P or di-hydro S-1-P.

[0020] In another embodiment, the method for identifying agonist orantagonist of a human EDG-1c polypeptide comprises:

[0021] determining the inhibition of binding of a ligand to cells whichhave the polypeptide on the surface thereof, or to cell membranescontaining the polypeptide, in the presence of a candidate compoundunder conditions to permit binding to the polypeptide, and determiningthe amount of ligand bound to the polypeptide, such that a compoundcapable of causing reduction of binding of a ligand is an agonist orantagonist. Preferably, the ligand is S-1-P or di-hydro S-1-P. Yet morepreferably, S-1-P or di-hydro S-1-P is labeled.

[0022] Furthermore, the present invention relates to treating conditionsassociated with human EDG-1c receptor imbalance with the identifiedcompounds. Yet another aspect of the invention relates to diagnosticassays for detecting diseases associated with inappropriate EDG-1activity or levels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 shows the nucleotide sequence of the human EDG-1c receptor(SEQ ID NO: 1).

[0024]FIG. 2 shows the deduced amino acid sequence of the human EDG-1creceptor (SEQ ID NO:2).

[0025]FIG. 3 shows concentration response curves for S-1-P againstendogeneous HEK293 cells.

[0026]FIG. 4 shows an agar plate assay of the dose response for S-1-Pagainst yeast cells containing the pathway-inducible fus1-lacZ reporterand expressing the human EDG-1c receptor in combination with either theendogenous yeast G? protein (GPA1) or a chimeric yeast G?/human G?i2.

[0027]FIG. 5 shows concentration response curves for S-1-P against yeastcells containing the pathway-inducible fus1-lacZ reporter and expressingthe human EDG-1c receptor in combination with GPA1 in a liquid lacZassay format.

[0028]FIG. 6 shows dose dependent cellular hypertrophy in rat neonatalmyocytes in culture induced by S-1-P.

[0029]FIG. 7 shows concentration-response curves for S-1-P in RBL 2H3cells stably transfected with the EDG-1c receptor.

[0030]FIG. 8 shows agonist activity for a number of ligands in RBL 2H3EDG-1c cells.

DESCRIPTION OF THE INVENTION Definitions

[0031] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0032] “Human EDG-1c” refers generally to polypeptides having the aminoacid sequence set forth in SEQ ID NO:2 or an allelic variant thereof.

[0033] “S-1-P (sphingosine-1-phosphate)” refers to the sphingolipidmetabolite having the structure:

[0034] “Di-hydro S-1-P” (di-hydro sphingosine-1-phosphate)” (hereinafterreferred to as “di-hydro S-1-P”) refers to the sphingolipid metabolitehaving the structure:

[0035] “Receptor Activity” or “Biological Activity of the Receptor”refers to the metabolic or physiologic function of said human EDG-1cincluding similar activities or improved activities or these activitieswith decreased undesirable side-effects. Also included are antigenic andimmunogenic activities of said human EDG-1c.

[0036] “Human EDG-1c polypeptides” refers to polypeptides with aminoacid sequences sufficiently similar to human EDG-1c preferablyexhibiting at least one biological activity of the receptor.

[0037] “Human EDG-1c gene” refers to a polynucleotide having thenucleotide sequence set forth in SEQ ID NO:1 or allelic variants thereofand/or their complements.

[0038] “Human EDG-1c polynucleotides” and refers to polynucleotidescontaining a nucleotide sequence which encodes a human EDG-1cpolypeptide of SEQ ID NO:2, or a nucleotide sequence which hassufficient identity to a nucleotide sequence contained in SEQ ID NO:1 tohybridize under conditions useable for amplification or for use as aprobe or marker.

[0039] “Antibodies,” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

[0040] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

[0041] “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0042] “Polypeptide” refers to any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres. “Polypeptide” refers to bothshort chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as posttranslational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan, et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

[0043] “Variant,” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

[0044] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLMNIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

[0045] Preferred parameters for polypeptide sequence comparison includethe following:

[0046] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0047] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992)

[0048] Gap Penalty: 12

[0049] Gap Length Penalty: 4

[0050] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0051] Preferred parameters for polynucleotide comparison include thefollowing:

[0052] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0053] Comparison matrix: matches=+10, mismatch=0

[0054] Gap Penalty: 50

[0055] Gap Length Penalty: 3

[0056] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0057] By way of example, a polynucleotide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO:1,that is be 100% identical, or it may include up to a certain integernumber of nucleotide alterations as compared to the reference sequence.Such alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleotide alterations is determined by multiplying the total number ofnucleotides in SEQ ID NO:1 by the numerical percent of the respectivepercent identity(divided by 100) and subtracting that product from saidtotal number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

[0058] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO:1, and y is, for instance,0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc., and wherein any non-integer product of x_(n) and y is rounded downto the nearest integer prior to subtracting it from x_(n). Alterationsof a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 maycreate nonsense, missense or frameshift mutations in this codingsequence and thereby alter the polypeptide encoded by the polynucleotidefollowing such alterations.

[0059] Similarly, a polypeptide sequence of the present invention may beidentical to the reference sequence of SEQ ID NO:2, that is be 100%identical, or it may include up to a certain integer number of aminoacid alterations as compared to the reference sequence such that the %identity is less than 100%. Such alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of amino acidalterations for a given % identity is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the numerical percent ofthe respective percent identity(divided by 100) and then subtractingthat product from said total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y)

[0060] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, and y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein anynon-integer product of x_(a) and y is rounded down to the nearestinteger prior to subtracting it from x_(a).

[0061] Polypeptides of the Invention

[0062] The human EDG-1c polypeptides of the present invention includethe polypeptide of SEQ ID NO:2 (in particular the mature polypeptide).

[0063] The human EDG-1c polypeptides may be in the form of the “mature”protein or may be a part of a larger protein such as a fusion protein.It is often advantageous to include an additional amino acid sequencewhich contains secretory or leader sequences, pro-sequences, sequenceswhich aid in purification such as multiple histidine residues, or anadditional sequence for stability during recombinant production.

[0064] Biologically active fragments of the human EDG-1c polypeptidesare also included in the invention. A fragment is a polypeptide havingan amino acid sequence that entirely is the same as part, but not all,of the amino acid sequence of the aforementioned human EDG-1cpolypeptides. As with human EDG-1c polypeptides, fragments may be“free-standing,” or comprised within a larger polypeptide of which theyform a part or region, most preferably as a single continuous region.Representative examples of polypeptide fragments of the invention,include, for example, fragments from about amino acid number 1-20,21-40, 41-60, 61-80, 81-100, and 101 to the end of human EDG-1cpolypeptides. In this context “about” includes the particularly recitedranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes.

[0065] Preferred fragments include, for example, truncation polypeptideshaving the amino acid sequence of human EDG-1c polypeptides, except fordeletion of a continuous series of residues that includes the aminoterminus, or a continuous series of residues that includes the carboxylterminus or deletion of two continuous series of residues, one includingthe amino terminus and one including the carboxyl terminus. Alsopreferred are fragments characterized by structural or functionalattributes such as fragments that comprise alpha-helix and alpha-helixforming regions, beta-sheet and beta-sheet-forming regions, turn andturn-forming regions, coil and coil-forming regions, hydrophilicregions, hydrophobic regions, alpha amphipathic regions, betaamphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions. Biologicallyactive fragments are those that mediate receptor activity, includingthose with a similar activity or an improved activity, or with adecreased undesirable activity. Also included are those that areantigenic or immunogenic in an animal, especially in a human.

[0066] Thus, the polypeptides of the invention include polypeptideshaving the amino acid sequence set forth in SEQ ID NO:2. Preferably, allof these polypeptides retain the biological activity of the receptor,including antigenic activity. Included in this group are variants of thedefined sequence and fragments. Preferred variants are those that varyfrom the referents by conservative amino acid substitutions—i.e., thosethat substitute a residue with another of like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, or 1-2amino acids are substituted, deleted, or added in any combination.

[0067] The human EDG-1c polypeptides of the invention can be prepared inany suitable manner. Such polypeptides include isolated naturallyoccurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. Means for preparing such polypeptides arewell understood in the art.

[0068] Polynucleotides of the Invention

[0069] Another aspect of the invention relates to isolatedpolynucleotides which encode the EDG-1 polypeptides and polynucleotidesclosely related thereto.

[0070] The nucleotide sequence of SEQ ID NO:1 shows homology with humanEDG-1 receptor (Hla, T., and T. Maciag; 1990; J. Biol. Chem. 265:9309-9313). The nucleotide sequence of SEQ ID NO:1 is a cDNA sequenceand comprises a polypeptide encoding sequence (nucleotide 1 to 1149)encoding a polypeptide of 382 amino acids, the polypeptide of SEQ IDNO:2. The nucleotide sequence encoding the polypeptide of SEQ ID NO:2may be identical to the polypeptide encoding sequence contained in SEQID NO:1 or it may be a sequence other than the one contained in SEQ IDNO:1, which, as a result of the redundancy (degeneracy) of the geneticcode, also encodes the polypeptide of SEQ ID NO:2. The polypeptide ofthe SEQ ID NO:2 is structurally related to other proteins of theG-coupled Protein Receptors family, having homology and/or structuralsimilarity with human EDG-1 receptor (Hla, T., and T. Maciag; 1990; J.Biol. Chem. 265: 9309-9313).

[0071] One polynucleotide of the present invention encoding human EDG-1cmay be obtained using standard cloning and screening, from a cDNAlibrary derived from mRNA in cells of human placenta using the expressedsequence tag (EST) analysis (Adams, M. D., et al. Science (1991)252:1651-1656; Adams, M. D. et al., Nature, (1992) 355:632-634; Adams,M. D., et al., Nature (1995) 377 Supp:3-174). Polynucleotides of theinvention can also be obtained from natural sources such as genomic DNAlibraries or can be synthesized using well known and commerciallyavailable techniques.

[0072] Thus, the nucleotide sequence encoding human EDG-1c polypeptidesmay be identical over its entire length to the coding sequence in FIG. 1(SEQ ID NO:1).

[0073] When the polynucleotides of the invention are used for therecombinant production of human EDG-1c polypeptide, the polynucleotidemay include the coding sequence for the mature polypeptide or a fragmentthereof, by itself; the coding sequence for the mature polypeptide orfragment in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other fusion peptide portions. For example, amarker sequence which facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz, et al., ProcNatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

[0074] Among particularly preferred embodiments of the invention arepolynucleotides encoding human EDG-1c polypeptides having the amino acidsequence of set out in FIG. 1 (SEQ ID NO:2) and variants thereof.

[0075] Further preferred embodiments are polynucleotides encoding humanEDG-1c variants that have the amino acid sequence of the human EDG-1c ofFIG. 1 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 aminoacid residues are substituted, deleted or added, in any combination.

[0076] The present invention further relates to polynucleotides thathybridize to the herein above-described sequences. In this regard, thepresent invention especially relates to polynucleotides which hybridizeunder stringent conditions to the herein above-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences.

[0077] Polynucleotides of the invention, which are sufficientlyidentical to a nucleotide sequence contained in SEQ ID NO:1, may be usedas hybridization probes for cDNA and genomic DNA, to isolate full-lengthcDNAs and genomic clones encoding human EDG-1c and to isolate cDNA andgenomic clones of other genes that have a high sequence similarity tothe human EDG-1c gene. Such hybridization techniques are known to thoseof skill in the art. Typically these nucleotide sequences are 70%identical, preferably 80% identical, more preferably 90% identical tothat of the reference sequence. The probes generally will comprise atleast 15 nucleotides. Preferably, such probes will have at least 30nucleotides and may have at least 50 nucleotides. Particularly preferredprobes will range between 30 and 50 nucleotides.

[0078] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments and diagnostics to animal and human disease.

[0079] Vectors, Host Cells, Expression

[0080] The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

[0081] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof forpolynucleotides of the present invention. Introduction ofpolynucleotides into host cells can be effected by methods described inmany standard laboratory manuals, such as Davis, et al., BASIC METHODSIN MOLECULAR BIOLOGY (1986) and Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989) such as calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction or infection.

[0082] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, E. coli, Streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

[0083] A great variety of expression systems can be used. Such systemsinclude, among others, chromosomal, episomal and virus-derived systems,e.g., vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook,et al., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

[0084] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0085] If the human EDG-1c polypeptide is to be expressed for use inscreening assays, generally, it is preferred that the polypeptide beproduced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If human EDG-1cpolypeptide is secreted into the medium, the medium can be recovered inorder to recover and purify the polypeptide; if producedintracellularly, the cells must first be lysed before the polypeptide isrecovered.

[0086] Human EDG-1c polypeptides can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

[0087] Diagnostic Assays

[0088] This invention also relates to the use of human EDG-1cpolynucleotides for use as diagnostic reagents. Detection of a mutatedform of human EDG-1c gene associated with a dysfunction will provide adiagnostic tool that can add to or define a diagnosis of a disease orsusceptibility to a disease which results from under-expression,over-expression or altered expression of human EDG-1c. Individualscarrying mutations in the human EDG-1c gene may be detected at the DNAlevel by a variety of techniques.

[0089] Nucleic acids for diagnosis may be obtained from a subject'scells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniquesprior to analysis. RNA or cDNA may also be used in similar fashion.Deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to labeled human EDG-1cnucleotide sequences. Perfectly matched sequences can be distinguishedfrom mismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing. See, e.g.,Myers, et al., Science (1985) 230:1242. Sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase and S1 protection or the chemical cleavage method. See Cotton, etal., Proc Natl Acad Sci USA (1985) 85: 4397-4401.

[0090] The diagnostic assays offer a process for diagnosing ordetermining a susceptibility to infections such as bacterial, fungal,protozoan and viral infections, particularly through detection ofmutation in the EDG-1 gene by the methods described.

[0091] In addition, infections such as bacterial, fungal, protozoan andviral infections, particularly infections such as bacterial, fungal,protozoan and viral infections, particularly infections caused by HIV-1or HIV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma;Parkinson's disease; acute heart failure; hypotension; hypertension;urinary retention; osteoporosis; angina pectoris; myocardial infarction;stroke; congestive heart failure; left ventricular hypertrophy;arrythmias; restenosis after coronary artery angioplasty; vascularsclerosis; deleterious fibrosis; atherosclerosis; inflammation;angiogenesis; wound healing; ulcers; asthma; allergies; benign prostatichypertrophy; migraine; vomiting; psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, depression,delirium, dementia, and severe mental retardation; degenerativediseases, such as neurodegenerative diseases and ischemic stroke; anddyskinesias, such as Huntington's disease or Gilles dela Tourett'ssyndrome, can be diagnosed by methods comprising determining from asample derived from a subject an abnormally decreased or increased levelof human EDG-1c polypeptide or human EDG-1c mRNA. Decreased or increasedexpression can be measured at the RNA level using any of the methodswell known in the art for the quantitation of polynucleotides, such as,for example, PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods. Assay techniques that can be used to determinelevels of a protein, such as human EDG-1c, in a sample derived from ahost are well-known to those of skill in the art. Such assay methodsinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays.

[0092] Chromosome Assays

[0093] The nucleotide sequences of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. The mapping of relevant sequences tochromosomes according to the present invention is an important firststep in correlating those sequences with gene associated disease. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

[0094] The differences in the cDNA or genomic sequence between affectedand unaffected individuals can also be determined. If a mutation isobserved in some or all of the affected individuals but not in anynormal individuals, then the mutation is likely to be the causativeagent of the disease.

[0095] Antibodies

[0096] The polypeptides of the invention or their fragments or analogsthereof, or cells expressing them can also be used as immunogens toproduce antibodies immunospecific for the human EDG-1c polypeptides. Theterm “immunospecific” means that the antibodies have substantiallygreater affinity for the polypeptides of the invention than theiraffinity for other related polypeptides in the prior art.

[0097] Antibodies generated against the human EDG-1c polypeptides can beobtained by administering the polypeptides or epitope-bearing fragments,analogs or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler, G. and Milstein,C., Nature (1975) 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor, et al., Immunology Today (1983) 4:72) andthe EBV-hybridoma technique (Cole, et al., MONOCLONAL ANTIBODIES ANDCANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

[0098] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can also be adapted to produce single chainantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms including other mammals, may be used to expresshumanized antibodies.

[0099] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or to purify the polypeptidesby affinity chromatography.

[0100] Antibodies against human EDG-1c polypeptides may also be employedto treat infections such as bacterial, fungal, protozoan and viralinfections, particularly infections such as bacterial, fungal, protozoanand viral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson'sdisease; acute heart failure; hypotension; hypertension; urinaryretention; osteoporosis; angina pectoris; myocardial infarction; stroke;congestive heart failure; left ventricular hypertrophy; arrythmias;restenosis after coronary artery angioplasty; vascular sclerosis;deleterious fibrosis; atherosclerosis; inflammation; angiogenesis; woundhealing; ulcers; asthma; allergies; benign prostatic hypertrophy;migraine; vomiting; psychotic and neurological disorders, includinganxiety, schizophrenia, manic depression, depression, delirium,dementia, and severe mental retardation; degenerative diseases, such asneurodegenerative diseases and ischemic stroke; and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome, among others.

[0101] Vaccines

[0102] Another aspect of the invention relates to a method for inducingan immunological response in a mammal which comprises inoculating themammal with human EDG-1c polypeptide, or a fragment thereof, adequate toproduce antibody and/or T cell immune response to protect said animalfrom infections such as bacterial, fungal, protozoan and viralinfections, particularly infections such as bacterial, fungal, protozoanand viral infections, particularly infections caused by HIV-1 or HIV-2;pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson'sdisease; acute heart failure; hypotension; hypertension; urinaryretention; osteoporosis; angina pectoris; myocardial infarction; stroke;congestive heart failure; left ventricular hypertrophy; arrythmias;restenosis after coronary artery angioplasty; vascular sclerosis;deleterious fibrosis; atherosclerosis; inflammation; angiogenesis; woundhealing; ulcers; asthma; allergies; benign prostatic hypertrophy;migraine; vomiting; psychotic and neurological disorders, includinganxiety, schizophrenia, manic depression, depression, delirium,dementia, and severe mental retardation; degenerative diseases, such asneurodegenerative diseases and ischemic stroke; and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome, among others.Yet another aspect of the invention relates to a method of inducingimmunological response in a mammal which comprises, delivering humanEDG-1c gene via a vector directing expression of human EDG-1cpolypeptide in vivo in order to induce such an immunological response toproduce antibody to protect said animal from diseases.

[0103] A further aspect of the invention relates to animmunological/vaccine formulation (composition) which, when introducedinto a mammalian host, induces an immunological response in that mammalto a human EDG-1c polypeptide wherein the composition comprises a humanEDG-1c polypeptide or human EDG-1c gene. The vaccine formulation mayfurther comprise a suitable carrier. Since human EDG-1c polypeptide maybe broken down in the stomach, it is preferably administeredparenterally (including subcutaneous, intramuscular, intravenous,intradermal etc. injection). Formulations suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats andsolutes which render the formulation isotonic with the blood of therecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

[0104] Screening Assays

[0105] The EDG1-c polypeptide of the present invention may be employedin a process for screening for compounds that bind to and activate theEDG1-c polypeptides of the present invention (called agonists), orinhibit the interaction of the EDG1-c polypeptides with receptor ligands(called antagonists). Thus, polypeptides of the invention may also beused to assess the binding of small molecule substrates and ligands in,for example, cells, cell-free preparations, chemical libraries, andnatural product mixtures. These substrates and ligands may be naturalsubstrates and ligands or may be structural or functional mimetics. SeeColigan, et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).

[0106] EDG1-c proteins are responsible for many biological functions,including many pathologies. Provided by the invention are screeningmethods to identify compounds and drugs that stimulate EDG1-c or thatinhibit the function or level of the polypeptide. In general, agonistsare employed for therapeutic and prophylactic purposes for suchconditions as infections such as bacterial, fungal, protozoan and viralinfections, particularly infections caused by HIV-1 or HIV-2; pain;cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson'sdisease; acute heart failure; hypotension; hypertension; urinaryretention; osteoporosis; angina pectoris; myocardial infarction; stroke;congestive heart failure; left ventricular hypertrophy; arrythmias;restenosis after coronary artery angioplasty; vascular sclerosis;deleterious fibrosis; atherosclerosis; inflammation; angiogenesis; woundhealing; ulcers; asthma; allergies; benign prostatic hypertrophy;migraine; vomiting; psychotic and neurological disorders, includinganxiety, schizophrenia, manic depression, depression, delirium,dementia, and severe mental retardation; degenerative diseases, such asneurodegenerative diseases and iscbemic stroke; and dyskinesias, such asHuntington's disease or Gilles dela Tourett's syndrome, among others.

[0107] In general, such screening procedures involve providingappropriate cells that express the receptor polypeptide of the presentinvention on the surface thereof. Such cells include cells from mammals,yeast, Drosophila or E. coli. In particular, a polynucleotide encodingthe receptor of the present invention is employed to transfect cells tothereby express the EDG1-c polypeptide. The expressed receptor is thencontacted with a test compound to observe binding, stimulation orinhibition of a functional response.

[0108] One such screening procedure involves the use of melanophoresthat are transfected to express the EDG1-c polypeptide of the presentinvention. Such a screening technique is described in PCT WO 92/01810,published Feb. 6, 1992. Such an assay may be employed to screen for acompound which inhibits activation of the receptor polypeptide of thepresent invention by contacting the melanophore cells that encode thereceptor with both a receptor ligand, such as S-1-P or di-hydro S-1-P,and a compound to be screened. Inhibition of the signal generated by theligand indicates that a compound is a potential antagonist for thereceptor, i.e., inhibits activation of the receptor.

[0109] The technique may also be employed for screening of compoundsthat activate the receptor by contacting such cells with compounds to bescreened and determining whether such compound generates a signal, i.e.,activates the receptor.

[0110] Other screening techniques include the use of cells which expressthe EDG1-c polypeptide (for example, transfected CHO cells) in a systemthat measures extracellular pH changes caused by receptor activation. Inthis technique, compounds may be contacted with cells expressing thereceptor polypeptide of the present invention. A second messengerresponse, e.g., signal transduction or pH changes, is then measured todetermine whether the potential compound activates or inhibits thereceptor.

[0111] Another screening technique involves expressing the EDG1-cpolypeptide in which the receptor is linked to phospholipase C or D.Representative examples of such cells include, but are not limited to:endothelial cells, smooth muscle cells, and embryonic kidney cells. Thescreening may be accomplished as hereinabove described by detectingactivation of the receptor or inhibition of activation of the receptorfrom the phospholipase second signal.

[0112] Another method involves screening for compounds that areantagonists, and thus inhibit activation of the receptor polypeptide ofthe present invention by determining inhibition of binding of labeledligand, such as S-1-P or di-hydro S-1-P, to cells expressing thereceptor on their surface, or cell membranes containing the receptor.Such a method involves transfecting a eukaryotic cell with DNA encodingthe EDG1-c polypeptide, such that the cell expresses the receptor on itssurface. The cell is then contacted with a potential antagonist in thepresence of a labeled form of a ligand, such as S-1-P or di-hydro S-1-P.The ligand can be labeled, e.g., by radioactivity. The amount of labeledligand bound to the receptors is measured, e.g., by measuringradioactivity associated with transfected cells or membrane from thesecells. If the compound binds to the receptor, the binding of labeledligand to the receptor is inhibited as determined by a reduction oflabeled ligand which binds to the receptors. This method is calledbinding assay. Naturally, this same technique can be used to look for anagonist.

[0113] The screening method may simply measure the binding of acandidate compound to the polypeptide, or to cells or membranes bearingthe polypeptide, or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound.Alternatively, the screening method may involve measuring or,qualitatively or quantitatively, detecting the competition of binding ofa candidate compound to the polypeptide with a labeled competitor (e.g.agonist or antagonist). Further, these screening methods may testwhether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide, using detection systemsappropriate to the cells bearing the polypeptide. Inhibitors ofactivation are generally assayed in the presence of a known agonist andthe effect on activation by the agonist by the presence of the candidatecompound is observed. Further, the screening methods may simply comprisethe steps of mixing a candidate compound with a solution containing apolypeptide of the present invention, to form a mixture, measuringEDG-1c activity in the mixture, and comparing the EDG-1c activity of themixture to a control mixture which contains no candidate compound.

[0114] Another screening procedure involves the use of mammalian cells(CHO, HEK 293, Xenopus Oocytes, RBL-2H3, etc.) that are transfected toexpress the receptor of interest. The cells are loaded with an indicatordye that produces a fluorescent signal when bound to calcium, and thecells are contacted with a test substance and a receptor agonist, suchas

[0115] S-1-P or di-hydro S-1-P. Any change in fluorescent signal ismeasured over a defined period of time using, for example, afluorescence spectrophotometer or a fluorescence imaging plate reader. Achange in the fluorescence signal pattern generated by the ligandindicates that a compound is a potential antagonist or agonist for thereceptor.

[0116] Another screening procedure involves use of mammalian cells (CHO,HEK293, Xenopus Oocytes, RBL-2H3, etc.) that are transfected to expressthe receptor of interest, and that are also transfected with a reportergene construct that is coupled to activation of the receptor (forexample, luciferase or beta-galactosidase behind an appropriatepromoter). The cells are contacted with a test substance and thereceptor agonist (ligand), such as S-1-P or di-hydro S-1-P, and thesignal produced by the reporter gene is measured after a defined periodof time. The signal can be measured using a luminometer,spectrophotometer, fluorimeter, or other such instrument appropriate forthe specific reporter construct used. Inhibition of the signal generatedby the ligand indicates that a compound is a potential antagonist forthe receptor.

[0117] Another screening technique for antagonists or agonists involvesintroducing RNA encoding the EDG1-c polypeptide into Xenopus oocytes (orCHO, HEK 293, RBL-2H3, etc.) to transiently or stably express thereceptor. The receptor oocytes are then contacted with the receptorligand, such as S-1-P or di-hydro S-1-P, and a compound to be screened.Inhibition or activation of the receptor is then determined by detectionof a signal, such as, cAMP, calcium, proton, or other ions.

[0118] Another method involves screening for EDG1-c polypeptideinhibitors by determining inhibition or stimulation of EDG1-cpolypeptide-mediated cAMP and/or adenylate cyclase accumulation ordimunition. Such a method involves transiently or stably transfecting aeukaryotic cell with EDG1-c polypeptide receptor to express the receptoron the cell surface. The cell is then exposed to potential antagonistsin the presence of EDG1-c polypeptide ligand, such as S-1-P or di-hydroS-1-P. The changes in levels of cAMP is then measured over a definedperiod of time, for example, by radioimmuno or protein binding assays(for example using Flashplates or a scintillation proximity assay).Changes in cAMP levels can also be determined by directly measuring theactivity of the enzyme, adenylyl cyclase, in broken cell preparations.If the potential antagonist binds the receptor, and thus inhibits EDG1-cpolypeptide-ligand binding, the levels of EDG1-c polypeptide-mediatedcAMP, or adenylate cyclase activity, will be reduced or increased.

[0119] Another screening method for agonists and antagonists relies onthe endogenous pheromone response pathway in the yeast, Saccharomycescerevisiae. Heterothallic strains of yeast can exist in two mitoticallystable haploid mating types, MATa and MATa. Each cell type secretes asmall peptide hormone that binds to a G-protein coupled receptor onopposite mating-type cells which triggers a MAP kinase cascade leadingto G1 arrest as a prelude to cell fusion. Genetic alteration of certaingenes in the pheromone response pathway can alter the normal response topheromone, and heterologous expression and coupling of human G-proteincoupled receptors and humanized G-protein subunits in yeast cells devoidof endogenous pheromone receptors can be linked to downstream signalingpathways and reporter genes (e.g., U.S. Pat. Nos. 5,063,154; 5,482,835;5,691,188). Such genetic alterations include, but are not limited to:(i) deletion of the STE2 or STE3 gene encoding the endogenous G-proteincoupled pheromone receptors; (ii) deletion of the FAR1 gene encoding aprotein that normally associates with cyclin-dependent kinases leadingto cell cycle arrest; and (iii) construction of reporter genes fused tothe FUS1 gene promoter (where FUS1 encodes a membrane-anchoredglycoprotein required for cell fusion). Downstream reporter genes canpermit either a positive growth selection (e.g., histidine prototrophyusing the FUS1-HIS3 reporter), or a colorimetric, fluorimetric orspectrophotometric readout, depending on the specific reporter constructused (e.g., b-galactosidase induction using a FUS1-LacZ reporter).

[0120] The yeast cells can be further engineered to express and secretesmall peptides from random peptide libraries, some of which can permitautocrine activation of heterologously expressed human (or mammalian)G-protein coupled receptors (Broach, et al., Nature 384: 14-16, 1996;Manfredi, et al., Mol. Cell. Biol. 16: 4700-4709, 1996). This provides arapid direct growth selection (e.g., using the FUS1-HIS3 reporter) forsurrogate peptide agonists that activate characterized or orphanreceptors. Alternatively, yeast cells that functionally express human(or mammalian) G-protein coupled receptors linked to a reporter genereadout (e.g., FUS1-LacZ) can be used as a platform for high-throughputscreening of known ligands, fractions of biological extracts andlibraries of chemical compounds for either natural or surrogate ligands.Functional agonists of sufficient potency (whether natural or surrogate)can be used as screening tools in yeast cell-based assays foridentifying G-protein coupled receptor antagonists. For example,agonists will promote growth of a cell with FUS-HIS3 reporter or givepositive readout for a cell with FUS1-LacZ. However, a candidatecompound that inhibits growth or negates the positive readout induced byan agonist is an antagonist. For this purpose, the yeast system offersadvantages over mammalian expression systems due to its ease of utilityand null receptor background (lack of endogenous G-protein coupledreceptors), which often interferes with the ability to identify agonistsor antagonists.

[0121] The present invention also provides a method for identifying newligands not known to be capable of binding to an EDG1-c polypeptide. Thescreening assays described above for identifying agonists may be used toidentify new ligands.

[0122] The present invention also contemplates agonists and antagonistsobtained from the above described screening methods.

[0123] Examples of potential EDG1-c polypeptide receptor antagonistsinclude peptidomimetics, synthetic organic molecules, natural products,antibodies, etc., that bind to the receptor but do not elicit a secondmessenger response, such that the activity of the receptor is prevented.

[0124] Potential antagonists also include proteins which are closelyrelated to the ligand of the EDG1-c polypeptide receptor, i.e., afragment of the ligand, which have lost biological function, and whenthey bind to the EDG1-c polypeptide receptor, elicit no response.

[0125] Thus in another aspect, the present invention relates to ascreening kit for identifying agonists, antagonists, and ligands forEDG1-c polypeptides, comprising:

[0126] (a) a EDG1-c polypeptide, preferably that of SEQ ID NO:2; andfurther preferably comprises labeled or unlabeled S-1-P or di-hydroS-1-P;

[0127] (b) a recombinant cell expressing a EDG1-c polypeptide,preferably that of SEQ ID NO:2; and further preferably comprises labeledor unlabeled S-1-P or di-hydro S-1-P; or

[0128] (c) a cell membrane expressing EDG1-c polypeptide; preferablythat of SEQ ID NO:2; and further preferably comprises labeled orunlabled S-1-P or di-hydro S-1-P.

[0129] It will be appreciated that in any such kit, (a), (b), or (c) maycomprise a substantial component.

[0130] As noted above, a potential antagonist is a small molecule whichbinds to the EDG1-c polypeptide receptor, making it inaccessible toligands such that normal biological activity is prevented. Examples ofsmall molecules include, but are not limited to, small peptides orpeptide-like molecules.

[0131] Potential antagonists also include soluble forms of EDG1-cpolypeptide receptor, e.g., fragments of the receptor, which bind to theligand and prevent the ligand from interacting with membrane boundEDG1-c polypeptide receptors.

[0132] The screening method may simply measure the binding of acandidate compound to the polypeptide, or to cells or membranes bearingthe polypeptide, or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound.Alternatively, the screening method may involve competition with alabeled competitor. Further, these screening methods may test whetherthe candidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells bearing the polypeptide. Inhibitors of activation aregenerally assayed in the presence of a known agonist and the effect onactivation by the agonist by the presence of the candidate compound isobserved. Constitutively active polypeptides may be employed inscreening methods for inverse agonists or inhibitors, in the absence ofan agonist or inhibitor, by testing whether the candidate compoundresults in inhibition of activation of the polypeptide. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution containing a polypeptide of the presentinvention, to form a mixture, measuring EDG-1c activity in the mixture,and comparing the EDG-1c activity of the mixture to a standard. Fusionproteins, such as those made from Fc portion and EDG-1c polypeptide, ashereinbefore described, can also be used for high-throughput screeningassays to identify antagonists for the polypeptide of the presentinvention (see D. Bennett et al., J. Mol. Recognition, 8:52-58 (1995);and K. Johanson et al., J. Biol. Chem., 270(16):9459-9471 (1995).

[0133] Polypeptides of the present invention may be employed inconventional low capacity screening methods and also in high-throughputscreening (HTS) formats. Such HTS formats include not only thewell-established use of 96- and, more recently, 384-well microtiterplates but also emerging methods such as the nanowell method describedby Schullek, et al., Anal Biochem., 246: 20-29 (1997).

[0134] Prophylactic and Therapeutic Methods

[0135] This invention provides methods of treating an abnormalconditions related to both an excess of and insufficient amounts ofhuman EDG-1c receptor activity.

[0136] If the activity of human EDG-1c receptor is in excess, severalapproaches are available. One approach comprises administering to asubject an inhibitor compound (antagonist) as hereinabove describedalong with a pharmaceutically acceptable carrier in an amount effectiveto inhibit activation by blocking binding of ligands to the human EDG-1creceptor, or by inhibiting a second signal, and thereby alleviating theabnormal condition.

[0137] In another approach, soluble forms of human EDG-1c polypeptidesstill capable of binding the ligand in competition with endogenous humanEDG-1c may be administered. Typical embodiments of such competitorscomprise fragments of the human EDG-1c polypeptide.

[0138] In still another approach, expression of the gene encodingendogenous human EDG-1c can be inhibited using expression blockingtechniques. Known such techniques involve the use of antisensesequences, either internally generated or separately administered. See,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied. See, for example,Lee, et al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science(1988) 241:456; Dervan, et al., Science (1991) 251:1360. These oligomerscan be administered per se or the relevant oligomers can be expressed invivo.

[0139] For treating abnormal conditions related to an under-expressionof human EDG-1c receptor and its activity, several approaches are alsoavailable. One approach comprises administering to a subject atherapeutically effective amount of a compound which activates humanEDG-1c receptor, i.e., an agonist as described above, in combinationwith a pharmaceutically acceptable carrier, to thereby alleviate theabnormal condition. Alternatively, gene therapy may be employed toeffect the endogenous production of human EDG-1c receptor by therelevant cells in the subject. For example, a polynucleotide of theinvention may be engineered for expression in a replication defectiveretroviral vector, as discussed above. The retroviral expressionconstruct may then be isolated and introduced into a packaging celltransduced with a retroviral plasmid vector containing RNA encoding apolypeptide of the present invention such that the packaging cell nowproduces infectious viral particles containing the gene of interest.These producer cells may be administered to a subject for engineeringcells in vivo and expression of the polypeptide in vivo. For overview ofgene therapy, see Chapter 20, Gene Therapy and other MolecularGenetic-based Therapeutic Approaches, (and references cited therein) inHuman Molecular Genetics, T Strachan and A P Read, BIOS ScientificPublishers Ltd. (1996).

[0140] Formulation and Administration

[0141] Peptides, such as the soluble form of human EDG-1c polypeptides,and agonists and antagonist peptides or small molecules, may beformulated in combination with a suitable pharmaceutical carrier. Suchformulations comprise a therapeutically effective amount of thepolypeptide or compound, and a pharmaceutically acceptable carrier orexcipient. Such carriers include but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. Formulation should suit the mode of administration, and is wellwithin the skill of the art. The invention further relates topharmaceutical packs and kits comprising one or more containers filledwith one or more of the ingredients of the aforementioned compositionsof the invention.

[0142] Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0143] Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

[0144] The dosage range required depends on the choice of peptide, theroute of administration, the nature of the formulation, the nature ofthe subject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

[0145] Polypeptides used in treatment can also be generated endogenouslyin the subject, in treatment modalities often referred to as “genetherapy” as described above. Thus, for example, cells from a subject maybe engineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

EXAMPLES Example 1 Yeast Cell Expression

[0146] The receptors of the present invention was constitutivelyexpressed in Saccharomyces cerevisiae using the PGK1 promoter carried ona standard 2-micron-based S. cerevisiae-E.coli shuttle plasmidcontaining the gene for ampillicin resistance, the ColE1 origin ofreplication and the S. cerevisiae LEU2 gene. The human EDG-1c cDNA wasmodified by trimming away the 5′ and 3′ UTRs and subcloned into theyeast expression vector. Following introduction into yeast cells usingstandard yeast genetic techniques, human EDG-1c polypeptide expressionwas detected by western blotting using a C-terminally tagged humanEDG-1c construct and antibodies to the epitope tag. Functionalexpression of human EDG-1c polypeptide (untagged) was determined asdescribed in Example 9.

Example 2 Ligand Bank for Binding and Functional Assays

[0147] A bank of over 600 putative receptor ligands has been assembledfor screening. The bank comprises: transmitters, hormones and chemokinesknown to act via a human seven transmembrane (7TM) receptor; naturallyoccurring compounds which may be putative agonists for a human 7TMreceptor, non-mammalian, biologically active peptides for which amammalian counterpart has not yet been identified; and compounds notfound in nature, but which activate 7TM receptors with unknown naturalligands. This bank is used to initially screen the receptor for knownligands, using both functional (i.e., calcium, cAMP, microphysiometer,oocyte electrophysiology, etc., see below) as well as binding assays.

Example 3 Ligand Binding Assays

[0148] Ligand binding assays provide a direct method for ascertainingreceptor pharmacology and are adaptable to a high throughput format. Thepurified ligand for a receptor is radiolabeled to high specific activity(50-2000 Ci/mmol) for binding studies. A determination is then made thatthe process of radiolabeling does not diminish the activity of theligand towards its receptor. Assay conditions for buffers, ions, pH andother modulators such as nucleotides are optimized to establish aworkable signal to noise ratio for both membrane and whole cell receptorsources. For these assays, specific receptor binding is defined as totalassociated radioactivity minus the radioactivity measured in thepresence of an excess of unlabeled competing ligand. Where possible,more than one competing ligand is used to define residual nonspecificbinding.

Example 4 Functional Assay in Xenopus Oocytes

[0149] Capped RNA transcripts from linearized plasmid templates encodingthe receptor cDNAs of the invention are synthesized in vitro with RNApolymerases in accordance with standard procedures. In vitro transcriptsare suspended in water at a final concentration of 0.2 mg/ml. Ovarianlobes are removed from adult female toads, Stage V defolliculatedoocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected ina 50 nl bolus using a microinjection apparatus. Two electrode voltageclamps are used to measure the currents from individual Xenopus oocytesin response to agonist exposure. Recordings are made in Ca2+ freeBarth's medium at room temperature. The Xenopus system can be used toscreen known ligands and tissue/cell extracts for activating ligands.

Example 5 Microphysiometric Assays

[0150] Activation of a wide variety of secondary messenger systemsresults in extrusion of small amounts of acid from a cell. The acidformed is largely as a result of the increased metabolic activityrequired to fuel the intracellular signaling process. The pH changes inthe media surrounding the cell are very small but are detectable by theCYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park,Calif.). The CYTOSENSOR is thus capable of detecting the activation of areceptor which is coupled to an energy utilizing intracellular signalingpathway such as the G-protein coupled receptor of the present invention.

Example 6 Extract/Cell Supernatant Screening

[0151] A large number of mammalian receptors exist for which thereremains, as yet, no cognate activating ligand (agonist). Thus, activeligands for these receptors may not be included within the ligands banksas identified to date. Accordingly, the 7TM receptor of the invention isalso functionally screened (using calcium, cAMP, microphysiometer,oocyte electrophysiology, etc., functional screens) against tissueextracts to identify natural ligands. Extracts that produce positivefunctional responses can be sequentially subfractionated until anactivating ligand is isolated identified.

Example 7 Calcium and cAMP Functional Assays

[0152] 7TM receptors which are expressed in HEK 293 cells have beenshown to be coupled functionally to activation of PLC and calciummobilization and/or cAMP stimulation or inhibition. Basal calcium levelsin the HEK 293 cells in receptor-transfected or vector control cellswere observed to be in the normal, 100 nM to 200 nM, range. HEK 293cells expressing recombinant receptors are loaded with fura 2 and in asingle day>150 selected ligands or tissue/cell extracts are evaluatedfor agonist induced calcium mobilization. Similarly, HEK 293 cellsexpressing recombinant receptors are evaluated for the stimulation orinhibition of cAMP production using standard cAMP quantitation assays.Agonists presenting a calcium transient or cAMP fluctuation are testedin vector control cells to determine if the response is unique to thetransfected cells expressing receptor.

Example 8 S-1-P-induced Ca2+ mobilization in untransfected HEK 293 cells

[0153] HEK 293 cells respond to S-1-P in a concentration-dependentmanner with a robust calcium mobilization response, indicating that thecells contain endogenous receptors that respond toS-1-P. FIG. 3 showsthe concentration response curves for S-1-P against untransfected HEK293 cells. The data were generated with the 96 well Fluorescent ImagingPlate Reader (FLIPR). Each point is the mean of 6-8 wells read on FLIPR.

Example 9 S-1-P-induced Reporter Gene Expression in Yeast

[0154] Human EDG-1c receptor was expressed in yeast strains containingendogenous yeast G-proteins and/or co-expressed yeast/human chimeric Gproteins, and/or human G-proteins. The yeast strain(s) used containmutations in genes in the pheromone response pathway, e.g., (i) deletionof the STE2 or STE3 gene encoding the endogenous G-protein coupledpheromone receptors; (ii) deletion of the FAR1 gene encoding a proteinthat normally associates with cyclin-dependent kinases leading to cellcycle arrest; and (iii) construction of reporter genes fused to the FUS1gene promoter (where FUS1 encodes a membrane-anchored glycoproteinrequired for cell fusion). The downstream reporter (FUS1-LacZ) permits acolorimetric or fluorimetric readout in response to ligand. FUS1-LacZcells expressing human EDG-1c demonstrated a receptor-dependent responseto S-1-P as determined by the expression of ?-galactosidase. Thisresponse, which is shown in FIG. 5, indicates functional coupling of thehuman EDG-1c receptor to yeast or yeast/human chimeric G-proteins.

Example 10 S-1-P-induced a Dose Dependent Cellular Hypertrophy in RatNeonatal Myocytes in Culture

[0155] S-1-P was tested in its ability to induce hypertrophy in an invitro neonatal cardiomyocyte model. The assessment of cardiomyocytehypertrophy is measured using four different parameters: proteinsynthesis (tritiated phenylalanine incorporation and protein contentincrease), tritiated thymidine incorporation (evaluation of fibroblastcontamination), Brain Natriuretic Peptide (BNP) release andmorphological parameters. Phenylephrine (PE) at 100 μM concentration isused as internal control experiments. S-1-P was applied at 10 nM, 100 nMand 1 μM (n=3). At each concentration S-1-P induced a cellularhypertrophy with an increase of protein content, phenylalanineincorporation and BNP secretion to the control cell values. FIG. 6 showsthe concentration response for S-1-P against rat neonatalcardiomyocytes. Cardiomyocytes in culture display features of myocytehypertrophy observed in vivo, such as changes in morphology (vizualisedusing light microscopy after staining with crystal violet), proteincontent, and pattern of gene expression. For example, at 1 μM (n=3),S-1-P induced a cellular hypertrophy with an 35.6%±6.3; 30.1%±9.2 and11.4%±1.8 increase of protein content, phenylalanine and thymidineincorporation respectively to the control cell values. BNP secretion wasfour fold higher in S-1-P treated cardiomyocyte vs. control.

Example 11 Human EDG1-c mRNA Expression in Human Cardiac Pathologies

[0156] Northern blot analysis was done either on 2 μg of poly A+ RNA ofeach sample fractionated on 1% formaldehyde-agarose gel, blotted on to anylon membrane (Hybond N+, Amersham) and subsequently hybridized usingstandard methods (Sambrook, et al., 1989) with a DNA fragment containingthe human EDG1-c gene. The DNA probe was labelled using (?-32P)-dCTP andthe Ready-prime labeling system (Amersham). Northern blots werehybridized overnight at 65° C. and subsequently washed with 0.1×SSC,0.1% SDS at 55° C. and exposed to X-ray film for 2-12 h. Northern blotexperiments on cardiac human pathological blot membranes indicatedreproducible overexpression of EDG-1 receptor mRNA in dilatedcardiomyopathy and ischemic samples.

Example 12 Functional Effects of S-1-P on Isolated Perfused Heart

[0157] The functional effects of S-1-P have been examined in isolatedperfused rabbit heart. S-1-P (10 nM) produced a slight negativeinotropic effect as the values were at 10 min of drug administration96.09±6 mm Hg and 84.7±6.3 mm Hg in controls versus S-1-P-treated group,respectively. An increased in AoP (aortic pressure), reflecting avasoconstrictor effect was observed, i.e., about 30% of increase at 5min of treatment compared to the vehicle (methanol 0.001%). A markedreduction of LVEDP (left ventricular end-diastolic pressure) was alsonoted.

Example 13 S-1-P- induced Calcium Mobilation Response in RBL 2H3 CellsStably Transfected with Human EDG-1c Receptor

[0158] Based on the results generated in FIG. 3, it was shown that anS-1-P exhibiteded an endogenous response in HEK 293 cells. A number ofcell lines were examined to identify one that would not respond to S-1-Pthrough an endogenous receptor. The cell line that we identified was RBL2H3 cells. Stable cell lines of the EDG1-c receptor were prepared in RBL2H3 cell line. The expression of functionally active clones werefollowed using Fluorescent imaging plate reader (FLIPR). The responsesfor several clones to S-1-P is presented in FIG. 7. As can be seen fromthis figure, the best clones respond in a concentration-dependent mannerwith EC₅₀s about 10-20 nM. The best clones were characterized further,and that is shown in FIG. 8. The cells responded with high potencythrough the EDG1-c receptor to S-1-P and dihydro-S-1-P with similarEC₅₀s in the 10-20 nM range and weakly to sphingosine phosphorylcholine(SPPC) with EC₅₀ in the uM range. The cells did not respond tolysophosphatidic acid (LPA, an EDG2 receptor ligand). Included in thefigure are the responses to endogenous receptors, namely, leukotriene D₄(LTD₄) and ATP to demonstrate that the cells were functionally in goodshape. These endogenous ligands gave the expected EC₅₀ values for thesecells. Muscarine and endogenous ligand for HEK 293, cells but not RBL2H3 cells, did not respond.

[0159] All publications including, but not limited to, patents andpatent applications, cited in this specification, are hereinincorporated by reference as if each individual publication werespecifically and individually indicated to be incorporated by referenceherein as though fully set forth.

[0160] The above description fully discloses the invention, includingpreferred embodiments thereof. Modifications and improvements of theembodiments specifically disclosed herein are within the scope of thefollowing claims. Without further elaboration, it is believed that oneskilled in the art can, using the preceding description, utilize thepresent invention to its fullest extent. Therefore, the examplesprovided herein are to be construed as merely illustrative and are not alimitation of the scope of the present invention in any way. Theembodiments of the invention in which an exclusive property or privilegeis claimed are defined as follows.

1 2 1 1149 DNA Human 1 atggggccca ccagcgtccc gctggtcaag gcccaccgcagctcggtctc tgactacgtc 60 aactatgata tcatcgtccg gcattacaac tacacgggaaagctgaatat cagcgcggac 120 aaggagaaca gcattaaact gacctcggtg gtgttcattctcatctgctg ctttatcatc 180 ctggagaaca tctttgtctt gctgaccatt tggaaaaccaagaaattcca ccgacccatg 240 tactatttta ttggcaatct ggccctctca gacctgttggcaggagtagc ctacacagct 300 aacctgctct tgtctggggc caccacctac aagctcactcccgcccagtg gtttctgcgg 360 gaagggagta tgtttgtggc cctgtcagcc tccgtgttcagtctcctcgc catcgccatt 420 gagcgctata tcacaatgct gaaaatgaaa ctccacaacgggagcaataa cttccgcctc 480 ttcctgctaa tcagcgcctg ctgggtcatc tccctcatcctgggtggcct gcctatcatg 540 ggctggaact gcatcagtgc gctgtccagc tgctccaccgtgctgccgct ctaccacaag 600 cactatatcc tcttctgcac cacggtcttc actctgcttctgctctccat cgtcattctg 660 tactgcagaa tctactcctt ggtcaggact cggagccgccgcctgacgtt ccgcaagaac 720 atttccaagg ccagccgcag ctctgagaag tcgctggcgctgctcaagac cgtaattatc 780 gtcctgagcg tcttcatcgc ctgctgggca ccgctcttcatcctgctcct gctggatgtg 840 ggctgcaagg tgaagacctg tgacatcctc ttcagagcggagtacttcct ggtgttagct 900 gtgctcaact ccggcaccaa ccccatcatt tacactctgaccaacaagga gatgcgtcgg 960 gccttcatcc ggatcatgtc ctgctgcaag tgcccgagcggagactctgc tggcaaattc 1020 aagcgaccca tcatcgccgg catggaattc agccgcagcaaatcggacaa ttcctcccac 1080 ccccagaaag acgaagggga caacccagag accattatgtcttctggaaa cgtcaactct 1140 tcttcctag 1149 2 382 PRT Human 2 Met Gly ProThr Ser Val Pro Leu Val Lys Ala His Arg Ser Ser Val 1 5 10 15 Ser AspTyr Val Asn Tyr Asp Ile Ile Val Arg His Tyr Asn Tyr Thr 20 25 30 Gly LysLeu Asn Ile Ser Ala Asp Lys Glu Asn Ser Ile Lys Leu Thr 35 40 45 Ser ValVal Phe Ile Leu Ile Cys Cys Phe Ile Ile Leu Glu Asn Ile 50 55 60 Phe ValLeu Leu Thr Ile Trp Lys Thr Lys Lys Phe His Arg Pro Met 65 70 75 80 TyrTyr Phe Ile Gly Asn Leu Ala Leu Ser Asp Leu Leu Ala Gly Val 85 90 95 AlaTyr Thr Ala Asn Leu Leu Leu Ser Gly Ala Thr Thr Tyr Lys Leu 100 105 110Thr Pro Ala Gln Trp Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu 115 120125 Ser Ala Ser Val Phe Ser Leu Leu Ala Ile Ala Ile Glu Arg Tyr Ile 130135 140 Thr Met Leu Lys Met Lys Leu His Asn Gly Ser Asn Asn Phe Arg Leu145 150 155 160 Phe Leu Leu Ile Ser Ala Cys Trp Val Ile Ser Leu Ile LeuGly Gly 165 170 175 Leu Pro Ile Met Gly Trp Asn Cys Ile Ser Ala Leu SerSer Cys Ser 180 185 190 Thr Val Leu Pro Leu Tyr His Lys His Tyr Ile LeuPhe Cys Thr Thr 195 200 205 Val Phe Thr Leu Leu Leu Leu Ser Ile Val IleLeu Tyr Cys Arg Ile 210 215 220 Tyr Ser Leu Val Arg Thr Arg Ser Arg ArgLeu Thr Phe Arg Lys Asn 225 230 235 240 Ile Ser Lys Ala Ser Arg Ser SerGlu Lys Ser Leu Ala Leu Leu Lys 245 250 255 Thr Val Ile Ile Val Leu SerVal Phe Ile Ala Cys Trp Ala Pro Leu 260 265 270 Phe Ile Leu Leu Leu LeuAsp Val Gly Cys Lys Val Lys Thr Cys Asp 275 280 285 Ile Leu Phe Arg AlaGlu Tyr Phe Leu Val Leu Ala Val Leu Asn Ser 290 295 300 Gly Thr Asn ProIle Ile Tyr Thr Leu Thr Asn Lys Glu Met Arg Arg 305 310 315 320 Ala PheIle Arg Ile Met Ser Cys Cys Lys Cys Pro Ser Gly Asp Ser 325 330 335 AlaGly Lys Phe Lys Arg Pro Ile Ile Ala Gly Met Glu Phe Ser Arg 340 345 350Ser Lys Ser Asp Asn Ser Ser His Pro Gln Lys Asp Glu Gly Asp Asn 355 360365 Pro Glu Thr Ile Met Ser Ser Gly Asn Val Asn Ser Ser Ser 370 375 380

What is claimed is:
 1. An isolated polynucleotide comprising anucleotide sequence encoding the polypeptide of SEQ ID NO:2; or anucleotide sequence complementary to said nucleotide sequence.
 2. Thepolynucleotide as claimed in claim 1, wherein said polynucleotide is DNAor RNA.
 3. The polynucleotide as claimed in claim 1, wherein saidnucleotide sequence comprises SEQ ID NO:1.
 4. An isolated polypeptidecomprising the polypeptide sequence set forth in SEQ ID NO:2.
 5. Anexpression system comprising a polynucleotide capable of producing apolypeptide as claimed in claim 4 when said expression system is in acompatible host cell.
 6. A process for producing a recombinant host cellcomprising the step of introducing the expression system as claimed inclaim 5 into a cell, such that the host cell, under appropriate cultureconditions, produces said polypeptide.
 7. A recombinant host cellproduced by the process as claimed in claim
 6. 8. A membrane of arecombinant host cell as claimed in claim 7 expressing said polypeptide.9. A process for producing a polypeptide comprising culturing a hostcell as claimed in claim 6 under conditions sufficient for theproduction of said polypeptide and recovering the polypeptide from theculture.
 10. An antibody immunospecific for the polypeptide as claimedin claim
 4. 11. A method for identifying agonist or antagonist of the ofpolypeptide as claimed in claim 4 comprising: (a) contacting a cellexpressing on the surface thereof the polypeptide, said polypeptidebeing associated with a second component capable of providing adetectable signal in response to the binding of a compound to saidpolypeptide, with a compound to be screened under conditions to permitbinding to the polypeptide; and (b) determining whether the compoundbinds to and activates or inhibits the polypeptide by measuring thelevel of a signal generated from the interaction of the compound withthe polypeptide.
 12. The method as claimed in claim 11, wherein saidmethod further comprises conducting the identification of an agonist orantagonist in the presence of labeled or unlabeled sphingosine1-phosphate or di-hydo sphingosine 1-phosphate.
 13. A method foridentifying an agonist or antagonist of the polypeptide as claimed inclaim 4 comprising: determining the inhibition of binding of a ligand tocells expressing the polypeptide on the surface thereof, or to cellmembranes containing the polypeptide, in the presence of a candidatecompound under conditions to permit binding to the polypeptide, anddetermining the amount of ligand bound to the polypeptide, such that acompound capable of causing reduction of binding of a ligand is anagonist or antagonist.
 14. The method as claimed in claim 13, whereinthe ligand is labeled or unlabeled sphingosine-1-phosphate or di-hydrosphingosine 1-phosphate.
 15. A method for screening to identifycompounds that stimulate or that inhibit a function or level of thepolypeptide as claimed in claim 4, comprising a method selected from thegroup consisting of: (a) measuring or, quantitatively or qualitatively,detecting the binding of a candidate compound to the polypeptide (or tothe cells or membranes bearing the polypeptide) or a fusion proteinthereof by means of a label directly or indirectly associated with thecandidate compound; (b) measuring the competition of the binding of acandidate compound to the polypeptide (or to the cells or membranesbearing the polypeptide) or a fusion protein thereof in the presence ofa labeled competitor, preferably sphingosine-1-phosphate or di-hydrosphingosine 1-phosphate; (c) testing whether the candidate compoundresults in a signal generated by activation or inhibition of thepolypeptide, using detection systems appropriate to the cells or cellmembranes bearing the polypeptide; (d) mixing a candidate compound witha solution comprising said polypeptide to form a mixture, measuringactivity of the polypeptide in the mixture, and comparing the activityof the mixture to a to a control mixture which contains no candidatecompound; or (e) detecting the effect of a candidate compound on theproduction of mRNA encoding said polypeptide and said polypeptide incells.
 16. An antagonist identified by the method as claimed in claim15.
 17. An agonist identified by the method as claimed in claim
 15. 18.A method for the treatment of a subject having need to inhibit activityor expression of human EDG-1c polypeptide comprising: (a) administeringto the subject a therapeutically effective amount of an antagonist asclaimed in claim
 16. 19. The method as claimed in claim 18, wherein thesubject is afflicted with a disease selected from the group consistingof: congestive heart failure, left ventricular hypertrophy, andarrythmias.